The Kinetics and Inhibition of Gamma-Glutamyl Transpeptidase A Biochemistry Laboratoty Experiment A. G. Spliigerber and Julie Sohl Gustavus Adolphus College, St. Peter, MN 56082 Most, if not all, published enzyme kinetics experiments involve single-substrate enzymes such as chymotrypsin ( I ) , lactase ( 2 ) ,or carhonic anhydrase (3).A laboratory experiment involving a two-substrate system might be a useful addition to the undergraduate biochemistry laboratory since this situation is probably more often encountered in nature. The use of a two-substrate system would demonstrate to the student not only the wider range of possible kinetic procedures but also that the degree of complexity of the data treatment is not much greater than in the single substrate case. A very versatile system, which may actually be used with either one or two substrates, involves the enzyme yglutamyl transpeptidase. The use of this enzyme in the laboratory might be rationalized by pointing out that the measurement of the level of this enzyme in blood serum is of some clinical significance. For example, elevated levels are indicative of liver disease (4, 5) and are frequently seen in alcoholism (6).Elevated levels are also found in neurological disorders such as epilepsy (7). Increased enzyme activity in the serum of patients with myocardial infarction may indicate reparative processes in the damaged tissue (8). The Blsubstrate Reactlon The normal two-substrate reaction, which for monitoring purposes utilizes the artificial substrate L-y-glutamyl-p-uitroauilide (GPN) and also uses a dipeptide such as glycylgycine (GG) as a second substrate, produces p-nitroaniline (PNA) as one product, with the y-glutamyl residue being transferred to the glycylglycine to form as the second product the tripeptide y-glutamylglycylglycine (GGG). Thus, GPN + GG
-
PNA + GGG
Theory of Bisubstrate Reactions There are two possible bisubstrate mechanisms, known respectively as sequential and "Ping Pong" mechanisms. In the notation due to Cleland (9), the "Ping Pong" mechanism may be written
where A and B are substrates and P and Q products. The various possible enzyme forms with bound substrate and product are also shown. Enzyme form F is an unbound species differing in some resuect such as oxidation state or conformation from form ~ . ' A l t e r n a t i naddition ~ of substrate and release of product is rharacteristic of this mechanism. Journal of Chemical Education
where [A] and [B] are substrate concentrations, K. and Kb are constants, and V,. is the maximum velocity a t high substrate levels. One of the reciprocal [Lineweaver-Burk] forms of the above equation is
This equation implies a linear plot of 1/V versus 1/[A] a t constant [B]. In this form of the equation, A is the variable substrate and B the "changing fixed substrate". A family of parallel lines results from changing the constant value of [B]. Parallel reciprocal plots are characteristic of a "Ping Pong" mechanism, The Cleland notation for the sequential mechanism is written
1
1
t
t
E
E
EA
EAB
(5)
EQ
In this mechanism both substrates are bound and then both products are released. The initial velocity equation for the sequential mechanism is given by
(1)
The yellow PNA product is easily monitored spectrophotometrically at 405 nm. The structural similarity of GGG to the naturally occurring tripeptide glutathione is worth a passing mention.
928
The initial velocity equation ([PI = [Q] = 0) for the "Ping Pong" mechanism is given by (10)
where Ki, is a constant not present in the "Ping Pong" equation. The reciprocal equation may be written as
The family of lines produced a t different fixed B levels converges to an intersection point to the left of the vertical axis. A family of converging lines is characteristic of a sequential mechanism. Procedure and Results The data that follow were collected from continuous assays using a Varian 620 spectrophotometer linked to a Sargent Model SR recorder. Velocities were measured from the slopes of plots of absorbance versus time. Alternatively, for large classes, a calorimeter such as aB&L Spectronic 20 may be easily substituted. In this case the assay is now a fixed time one with the reaction quenched by addition of acid after a convenient time interval.
1/ CGPNI
-
-
Figure 1 Plots of raclproca velocity versJs reclprocal mo ar GGconcensatm at I xed IGPN] Velocnty plonad as absorbance change per mmute 0.[GPNI = 0 0033 M A. [GPNJ = 0 0017 M. 0. IGPNJ = 0 00067 M
FImm 3. Interce0!3 from Fbwe 1 versus recl~rocal mllllmlar GPN concenbation. Intercepts plotted as reciprocal absorbance change per minute. Calculated Kb = 2.3 X loe3 M.
Figure 2. Plotsof reciprocal velocllyversus reclprocal millimolar GPN concentration at fixed [GG]. Velocity plotted as absorbance change per minute. 0, [GG] = 0.033 M; A. [GG] = 0.017 M; 0. [GGJ = 0.067 M.
Figure 4. lmercepts from Figure 2 versus reciprocal GG molarity. Intercepts plotted as reciprocal absorbance change per minute. Calculated 6 = 6.7 X
Reactions were carried out in 0.1 M phosphate buffer, pH 8.5. Stock GG and GPN (Sigma Chemical Co., St. Louis, MO) solutions were made up in buffer to 0.1066 M and 0.01066 M, respectively, the GPN solution being heated slightly to facilitate dissolution. Nonenzymatic hydrolysis of GPN is noticeable but occurs too slowly to affect the kinetic results. In each assay mixture 0.2 mL of astock 4.0 units/mL enzyme (Sigma) solution was used. Amounts of stock GG and GPN were varied from 0 to 1.5 mL and the total volume of the assay mixture adjusted to 3.2 mL with buffer. This volume is convenient either for 1-cm quartz cuvettes or for the round colorimeter cuvettes. If desired, Michaelis plots of velocity versus [GPN] or lGGl mav" he constructed to show the hvoerbolic nature of ;he curves with respect to both substra~s:If these plots are made, convenient substrate ranges are from 0 to 1.5 mL (0 to ) a constant 1-mL 0.05 M for GG, 0 to 0.005 M ~ O I G P N with amount of the fixed substrate (0.033 M for GG, 0.0033 M for
GPN). Neither plot shows any apparent sigmoid character. For purposes of identifying the type of reaction mechanism, Lineweaver-Burk (L-B) plots were made with respect to both substrates. The first set of plots (Fig. I), has GG as the variable substrate (from 0.33 mL to 1.0 mL or 0.011 M to 0.033 M) a t three different constant GPN levels (0.00067 M to 0.0033 M). The three parallel lines indicate a"Ping Pong" mechanism. The same mechanism is also indicated (Fia. 2) by L-B plots using GPN as the variable substrate (0.0011 M 10 0.01J33 M)at three constant CG level3 10.0067 % to 0.003 I
.
1 0 P M.
~~~~
~
Kinetic Constants
From eq 4, denoting GG as substrate A and GPN as suhstrate B, the L-B plots with GG as the variable substrate will have intercepts on the l l V axis of (l/Vm,,)(l KbI[GPNl). The kinetic constant Kb may be found by plotting the intercepts from the family of L-B plots of Figure 1 versus 11
+
Volume 65
Number 10
October 1986
929
I
I
.6
.3 Figure 5. Plots of reolprocal velocity versus reciprocal 00 molarity at fixed [GPN] wlm (A) and wimout ( 0 ) citrate Inhibitor. [GPN] = 0.0033 M and [citrate] = 0.016 M where present.
0
Figure 7. Absmbdnce versus time plots for the double-substrate system (A) and the single-subsham system (0). I&ntical conditions maintained for both reactions except far absence of GG in single-substratecase.
[GPN]. The intercept of this secondary plot (Fig. 3) is 1/Vand theslope id KdV,,,.,, from which Kb may be found. From the L-B plots with GPN as the variable substrate, K. may be found (Fig. 4) similarly by plotting the intercepts versus 11 [GGI. lnhibltor Studles I t has been reported (11)that fluoride, oxalate, and citrate ions inhibit the enzyme. However, only the citrate ion has a ~ronouncedeffect on reaction rate. L-B plots with one suhitrate level fixed and with constant inhibitor level may be inhibition with respect toeach used todetermine the typeof .. substrate. The plots (Fig. 5) with GG as variable substrate (0.011 M to 0.033 M) a t constant GPN level (0.0033 M) and either no inhibitor or a fixed inhibitor level of 0.016 M (0.5 mL of 0.1 M sodium citrate) shows com~etitiveinhibition with respect i nthe ~ 1tVaxis). With variable CPN to GG (lines ~ o n v & ~at (O.OU11 M to0.0033 M)and fixed GG 10.033 M Ithe inhibitor Journal of Chemical Education
Figure 6 Plots of reclpmcal velocity versus reopmcal m~lllmalarGPN wncenhallon at fixed (GG] wnh (Ajand wnhout(0)c haleinhob tor [GG]= 0 033 M and [citrate] = 0.016 M where present.
I
260 400 600 TIME, MINUTES
930
I
1.2
1/ CGPNI
1 / CGGI
I
I
.9
Figwe 8. Rots of reciprocal velocity vsrss reclprocal mil imolar WN conceh Dalton n the aosence of 00 wilh ( A and 01and witho~t( 0 ) 0.0 16 M cihate inhibitor.
present (0.016 M) and no-inhibitor plots converge to the left of the l l V axis. showine noncom~etitiveinhibition (Firr. 6). This inhibition' patter~(compet~ive-noncompetitive)does not conform to anv of the listed ~ossihilities(12) for "Ping Pong" bi bi kinetLcs for cases where the inhibitor binds to only one form of the enzyme. Therefore, for this particular enzyme, the inhibitor may hind to more than one form (E, E A P P , F, or FBfEQ) of the enzyme. Slngle-Substrate Kinetics In addition to the normal two-substrate transferase mechanism. two other sinde-substrate mechanisms involvine the .~~~~~ ~~~, substrate containinithe glutamyl residue have been reported ( 4 ) . A transferase mechanism mav occur which uses G P N as both donor and acceptor: ~
~
~
~
~
GPN + GPN
-
PNA + GGPN
(8)
In this case one would expect that only half the potential
PNA would beliberated. Apparently, the enzyme alsoacts to catalyze the hydrolysis of GPN: H 2 0 + G P N - G t P N A
(9)
This hydrolytic cleavage would result in the eventual liheration of all the potential PNA. A comparison of the amounts of PNA produced with time (Fig. 7) in the two-substrate and one-substrate cases shows not only that the single-suhsrrate reaction is much slower than the two-substrate one but also that all or nearly all of the potential PNA is liberated in either case if the reactions are allowed to proceed to completion. This would indicate hut not prove that the hydrolytic single-substrate merhanism predominates. The GGPN product of the single suhstrate transferase reaction mieht also be readilv"hvdrolvzed " . " to liberate PNA. A Micbaelis plot (V versus [S]) for the single-substrate reaction may be obtained by the student if desired. I t shows the usual hyperbolic behavior. Inhibition by citrate ion is still evident. L-B plots withand without inhibitor are shown in Figure 8 and indicate competitive inhibition. This contrasts with the behavior in the presence of both substrates, where the inhibition was noncompetitive with respect to GPN. Conclusion
An advantage of this particular enzyme system is that the absorbance-versus-time curve remains linear for a relatively long period of time (see Fig. 7). Tbis allows the student to measure accurately the velocity from the slope of the trace. I t also allows a fixed time assay to be conveniently used with a colorimeter such as the B&L Spectronic 20. With the fixed time assay the reaction is simply stopped after two minutes by addition to 0.5 mL of 30% acetic acid. A blank made up without enzyme is necessary for the colorimeter experiments. The major expense is the lyophilized enzyme, but the cost is not prohibitive since sufficient reagent for 10 pairs of
students would be approximately $26.00. If the enzyme cost is considered excessive. a small amount of human blood plasma may be substituted. If Michaelis d o t s are not rewired, the data can he collected in one f o u ~ b o u rlaboratory period. This assumes plots consistine of five data points per line and two or three lines per plot. Tbis experiment adds another dimension to the usual enzyme kinetics laboratory, exposing the student to procedures used to determine the type of enzyme mechanism, the kinetic constants, and the inhibition patterns. If desired the students may consult the literature (12) and try to determine the enzyme forms that are being complexed by the inhibitor. I t is also possible to simulate two-substrate enzyme kinetics with a computer. A three-part program written in the BASIC language is available from the authors on request. I t uses a random number generator to determine the type of mechanism ("Ping Pong" or sequential) and the type of inhibition pattern, assuming the inhibitor binds to only one form of the enzyme. There are three possible patterns for sequential mechanisms and three for the "Ping Pong" mechanism. The program will provide substrate concentrationvelocity data, line slopes a t various substrate concentrations, or families of L-B plots, depending on the section of the program being run. ~
~~
Literature Cited 1. 2. 3. 4.
5.
M.L.: Keady, F. J.;Wedler, F.C. J.Chem. Educ 1967,44,84 Russo, S. F.; Moothart, L. J,Chsm. Edur. 1986.62.242. Spyridia. G. T.;Meany, J. E. J. C h ~ mEduc . 1985.62,1125. Szezeklik.E.;Orlowski, M.;Szouezuk,A.Goslrnrnferology 1961.41.353. Bender,
Gambino,S.R.:Lum, G. Clin Chem. 1971,17,641.
6. Kri~ten~on,H.:Trell,E.;Eriksson, S.: Hanik, L.:Hood, B.Lones1 1977.1,609. 7. Ewen,L.M.;Grillitha,S.Clin.Cham. 1971,17,€42. 8. Revenn, K. G.; Gudbjamason, S.: Cowan.C. M . ; Bing, R. J. Circulation 1069.39.693. 9. Cleland, W . W . Riochim. Biophya. Acfo 1963,67,104. 10. C. J,Phys Chrm. 1956.60.1373. 11. Wolf, P. L.; Williams, D.; VonderMuehl. E. Procfirol Clinical Eniymoiogy and Bio
